Figure 2. Hybridoma production of monoclonal antibodies.

The Functional Properties of Antibodies BE 4332 Adapted from Molecular Biology of the Cell 4th ed., 1994, by Alberts et al., and Immunology, 4th ed, 2000, by Goldsby et al.

Antibodies defend us against infection by inactivating viruses and bacterial toxins and by recruiting the complement system and various types of white blood cells to kill extracellular microorganisms and larger parasites. Synthesized exclusively by B cells, antibodies are produced in millions of forms, each with a different amino acid sequence and a different binding site for antigen. Collectively called immunoglobulins (abbreviated as Ig), they are among the most abundant protein components in the blood, constituting about 20% of the total plasma protein by weight.

The Antigen-specific Receptors on B Cells Are Antibody Molecules. All antibody molecules made by an individual B cell have the same antigen-binding site. The first antibodies made by a newly formed B cell are not secreted. Instead, they are inserted into the plasma membrane, where they serve as receptors for antigen. Each B cell has approximately 105 such antibody molecules in its plasma membrane. Each of these antibody molecules is noncovalently associated with an invariant set of transmembrane polypeptide chains that are involved in passing signals to the cell interior when the extracellular binding site of the antigen is occupied by antigen.

When a virgin or a memory B cell is activated by antigen (with the aid of helper T cells), it proliferates and matures to become an antibody-secreting cell. The activated cells make and secrete large amounts of soluble (rather than membrane-bound) antibody, which has the same unique antigen-binding site as the cell-surface antibody that served earlier as the antigen receptor (Figure 1). Activated B cells can begin secreting antibody while they are still small lymphocytes, but the end stage of their maturation pathway is a large plasma cell, which secretes antibodies at the rapid rate of about 2000 molecules per second. Plasma cells seem to have committed so much of their protein-synthesizing machinery to making antibody that they are incapable of further growth and division, and most die after several days.

Figure 1. B cell activation. When resting B cells are activated by antigen to proliferate and mature into antibody-secreting cells, they produce and secrete antibodies with a unique antigen-binding site, which is the same as that of their original membrane-bound antibodies that served as antigen receptors.

B Cells Can Be Stimulated to Secrete Antibodies in a Culture Dish. In the 1960s it was discovered that B cells could be induced to secrete antibodies by exposing them to antigen in a test tube or cell culture dish, where cell-cell interactions can be manipulated and the environment controlled. The monoclonal antibody industry grew from this fact and a technique known as hybridoma fusion. Detailed at the figure to the left, large amounts of antibody are produced from a hybrid cell fused from a lymphocyte and a myeloma (tumor) cell.

Antibodies Have Two Identical Antigen-binding Sites. The simplest antibodies are Y-shaped molecules with two identical antigen-binding sites, one at the tip of each arm of the Y. Because of their two antigen-binding sites, they are said to be bivalent . As long as an antigen has three or more antigenic determinants, bivalent antibody molecules can cross-link it into a large lattice, which can be rapidly phagocytosed and degraded by macrophages. The efficiency of antigen binding and cross-linking is greatly increased by a flexible hinge region in antibodies, which allows the distance between the two antigen-binding sites to vary (Figure 3).

An Antibody Molecule Is Composed of Two Identical Light Chains and Two Identical Heavy Chains. The basic structural unit of an antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The four chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with the same antigen-binding site, and both light and heavy chains usually cooperate to form the antigen-binding surface (Figure 3).

There Are Five Classes of Heavy Chains, Each with Different Biological Properties. In higher vertebrates there are five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain - a, d, e, g, and m, respectively. IgA molecules have a chains, IgG molecules have g chains, and so on. In addition, there are a number of subclasses of IgG and IgA immunoglobulins; for example, there are four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) having g1 , g2 , g3 , and g4 heavy chains, respectively. The various heavy chains impart a distinctive conformation to the hinge and tail regions of antibodies and give each class (and subclass) characteristic properties of its own.

Antibodies Can Have Either k or l Light Chains, but Not Both. In addition to the five classes of heavy chains, higher vertebrates have two types of light chains, k and l, either of which may be associated with any of the heavy chains. An individual antibody molecule always consists of identical light chains and identical heavy chains; therefore, its two antigen-binding sites are always identical. This symmetry is crucial for the cross-linking function of secreted antibodies. An Ig molecule, consequently, may have either k or l light chains, but not both. No difference in the biological function of these two types of light chain has yet been identified.

The Strength of an Antibody-Antigen Interaction Depends on Both the Number of Antigen-binding Sites Occupied and the Affinity of Each Binding Site. The binding of an antigen to antibody, like the binding of a substrate to an enzyme, is reversible. It is mediated by the sum of many relatively weak noncovalent forces, including hydrophobic and hydrogen bonds, van dweak forces are effective only when the antigen molecule is close enoucomplementary recesses on the surface of the antibody. The complemetwo identical antigen-binding sites; the corresponding region on the an

Figure 3. A schematic drawing of a typical antibody molecule . It is composed of two identical heavy chains and two identical light chains. Note that the antigen-binding sites are formed by a complex of the amino-terminal regions of both light and heavy chains, but the tail and hinge regions are formed by the heavy chains alone.

Figure 4 . Antigen binding to antibody . In this highly schematized diagram, an antigenic determinant on a macromolecule is shown interacting with the antigen-binding site of two different antibody molecules, one of high and one of low affinity. The antigenic determinant is held in the binding site by various weak noncovalent forces, and the site with the better fit to the antigen has a greater affinity. Note that both the light and heavy chains of the antibody molecule usually contribute to the antigen-binding site.

er Waals forces, and ionic interactions. These gh to allow some of its atoms to fit into ntary regions of a four-chain antibody unit are its tigen is an antigenic determinant (Figure 4).

Figure 5 . Molecules with multiple antigenic determinants . (A) A globular protein with a number of different antigenic determinants. Different regions of a polypeptide chain usually come together in the folded structure to form each antigenic determinant on the surface of the protein. (B) A polymeric structure with many identical antigenic determinants; such a molecule is called a multivalent antigen.

Most antigenic macromolecules have many different antigenic determinants; if two or more of them are identical (as in a polymer with a repeating structure), the antigen is said to be multivalent (Figure 5.)

The reversible binding reaction between an antigen with a single antigenic determinant (denoted Ag) and a single antigen-binding site (denoted Ab) can be expressed as: AgAbAbAg ⇔+

The equilibrium point depends both on the concentrations of Ab and Ag and on the strength of their interaction. Clearly, a larger fraction of Ab will become associated with Ag as the concentration of Ag is increased. The strength of the interaction is generally expressed as the affinity constant ( Ka ), where (the square brackets indicate the concentration of each component at

equilibrium.)[ ][ ][ ]AbAg

AgAbK a =

The affinity constant, sometimes called the association constant, can be determined by measuring the concentration of free Ag required to fill half of the antigen-binding sites on the antibody. When half the sites are filled, [AgAb] = [Ab] and Ka = 1/[Ag]. Thus the reciprocal of the antigen concentration that produces half-maximal binding is equal to the affinity constant of the antibody for the antigen. Common values range from as low as 5 x 104 to as high as 1011

liters/mole. The affinity constant at which an immunoglobulin molecule ceases to be considered an antibody for a particular antigen is somewhat arbitrary, but it is unlikely that an antibody with a Ka below 104 would be biologically effective; moreover, B cells with receptors that have such a low affinity for an antigen are unlikely to be activated by the antigen.

Figure 6. Review of equilibrium. The equilibrium between molecules A and B and the complex AB is maintained by a balance between the two opposing reactions shown in (1) and (2). As shown in (3), the ratio of the rate constants for the association and the dissociation reactions is equal to the equilibrium constant (K) for the reaction. Molecules A and B must collide in order to react, and the rate in reaction (2) is therefore proportional to the product of their individual concentrations. As a result, the product [A] x [B] appears in the final expression for K, where [ ] indicates concentration. As traditionally defined, the concentrations of products appear in the numerator and the concentrations of reactants appear in the denominator of the equation for an equilibrium constant. Thus the equilibrium constant in (3) is that for the association reaction A + B AB. For simple binding interactions this constant is called the affinity constant or association constant (in units of liters per mole); the larger the value of the association constant ( K a), the stronger is the binding between A and B. The reciprocal of K ais the dissociation constant (in units of moles per liter); the smaller the value of the dissociation constant ( K d), the stronger is the binding between A and B.

The affinity of an antibody for an antigenic determinant describes the strength of binding of a single copy of the antigenic determinant to a single antigen-binding site, and it is independent of the number of sites. When, however, an antigen carrying multiple copies of the same antigenic determinant combines with a multivalent antibody, the binding strength is greatly increased because all of the antigen-antibody bonds must be broken simultaneously before the antigen and antibody can dissociate. Thus a typical IgG molecule can bind at least 50-100 times more strongly to a multivalent antigen if both antigen-binding sites are engaged than if only one site is engaged. The total binding strength of a multivalent antibody with a multivalent antigen is referred to as the avidity of the interaction.

Quantitative measurements of the affinity of an antibody for antigen can provide useful information about an antibody. For example, affinity measurements may be used to screen different isolates of an antibody in order to identify those that are most effective at binding antigen. Also, quantitative measurements of the capacity of an antibody to bind other compounds that are structurally related to the original immunizing antigen can help establish the likelihood of whether an antibody will crossreact, perhaps undesirably, with other molecules that might accompany the antigen. The valence of an antibody for antigen can also be found by quantitative affinity measurements, this parameter being an important distinguishing feature of different classes and subclasses of antibodies.

The simplest and most direct way of measuring antibody affinity is by the method of equilibrium dialysis:

(a) The dialysis chamber contains two compartments (A and B) separated by a semipermeable membrane. Antibody is added to one compartment and a radiolabeled ligand to another. At equilibrium the concentration of radioactivity in both compartments is measured. (b) Plot of concentration of ligand in each compartment with time. At equilibrium the difference in the concentration of radioactive ligand in the two compartments represents the amount of ligand bound to antibody.

From measurements of the equilibrium concentrations of free and bound antigen, starting with different initial concentrations of antigen, one can apply a simple formula in order to determine the equilibrium association constant and valence of an antibody. The formula is the Scatchard equation: r/c = K(n-r):

• r = moles bound ligand/mole antibody at equilibrium; • c = free ligand concentration at equilibrium; • K = equilibrium association constant; and • n = number of antigen binding sites per antibody molecule

By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis thus producing a Scatchard plot, as shown in Figure 6-3.

Scatchard plots are based on repeated equilibrium dialyses with a constant concentration of antibody and varying concentration of ligand. In these plots, r = moles bound ligand/mole antibody and c = free ligand. From a Scatchard plot, both the equilibrium constant (K) and the number of binding sites per antibody molecule (n), or its valency, can be obtained. (a) If all antibodies have the same affinity, then a Scatchard plot yields a straight line with a slope of -K. The Y intercept is the valence of the antibody, which is 2 for IgG. In this graph antibody #1 has a higher affinity than antibody #2. (b) If the antibodies are pooled and have a range of affinities, a Scatchard plot yields a curved line, whose slope is constantly changing. The average affinity constant K0 can be calculated by determining the value of K when one-half of the binding sites are occupied (i.e., when r = 1). In this graph antiserum #3 has a higher affinity (K0 = 2.4 x 108) than antiserum #4 (K0 = 1.25 x 108). If the equilibrium reaction relates to a monoclonal antibody, the Scatchard plot will yield a straight line, as illustrated in Figure 6-3(a). In this case the equilibrium association constant for antigen, K, equals the negative slope of this line and the antibody's valence, n, or number of antigen binding sites, equals the r value at the point where the extrapolated graphical line intercepts the X-axis. If the equilibrium reaction relates to a heterogeneous mixture of two or more antibodies recognizing the same antigen, the Scatchard plot will generally yield a curved line, as illustrated in Figure 6-3(b). In this case, the shape of the line depends on the molar ratio of different antibodies as well as their individual association constants. By definition, the average affinity, K0 = 1/c1/2, the reciprocal of the concentration of free antigen when one-half of the antigen binding sites are occupied. The average valence equals the r value at the point where the extrapolated graphical line intercepts the X-axis.

Biosensor assays for measuring the rates of association and disassociation of antigen receptors for their ligands. Two of the important questions that are always asked of any receptor-ligand interaction is: what is the strength of binding, or affinity, of the interaction, and what are the rates of association and disassociation? Traditionally, measurements of affinity have been made by equilibrium binding measurements, and measurements of rates of binding have been difficult to obtain. Equilibrium binding assays also cannot be performed on T-cell receptors, which have large macromolecular ligands and which cannot be isolated and purified in large quantity.

It is now possible to measure binding rates directly, by following the binding of ligands to receptors immobilized on gold-plated glass slides, using a phenomenon known as surface plasmon resonance to detect the binding. A full explanation of surface plasmon resonance is beyond the scope of this textbook, as it is based on advanced physical and quantum mechanical principles. Briefly, it relies on the total internal reflection of a beam of light from the surface of a gold-coated glass slide. As the light is reflected, some of its energy excites electrons in the gold coating and these excited electrons are in turn affected by the electric field of any molecules binding to the surface of the glass coating. The more molecules that bind to the surface, the greater the effect on the excited electrons, and this in turn affects the reflected light beam. The reflected light thus becomes a sensitive measure of the number of atoms bound to the gold surface of the slide.

If a purified receptor is immobilized on the surface of the gold-coated glass slide, to make a biosensor 'chip,' and a solution containing the ligand is flowed over that surface, the binding of ligand to the receptor can be followed until it reaches equilibrium. If the ligand is then washed out, dissociation of ligand from the receptor can easily be followed and the dissociation rate calculated. A new solution of the ligand at a different concentration can then be flowed over the chip and the binding once again measured. The affinity of binding can be calculated in a number of ways in this type of assay. Most simply, the ratio of the rates of association and dissociation will give an estimate of the affinity, but more accurate estimates can be obtained from the measurements of the binding at different concentrations of ligand. From measurements of binding at equilibrium, a Scatchard plot will give a measurement of the affinity of the receptor-ligand interaction.

Measurement of receptor-ligand interactions can be made in real time using a biosensor. Biosensors are able to measure the binding of molecules on the surface of gold-plated glass chips through the indirect effects of the binding on the total internal reflection of a beam of polarized light at the surface of the chip. Changes in the angle and intensity of the reflected beam are measured in 'resonance units' (Ru) and plotted against time in what is termed a 'sensorgram.' Depending on the exact nature of the receptor-ligand pair to be analyzed, either the receptor or the ligand can be immobilized on the surface of the chip. In the example shown, MHC:peptide complexes are immobilized on such a surface (first panel). T-cell receptors in solution are now allowed to flow over the surface, and to bind to the immobilized MHC:peptide complexes (second panel). As the T-cell receptors bind, the sensorgram (inset panel below the main panel) reflects the increasing amount of protein bound. As the binding reaches either saturation or equilibrium (third panel), the sensorgram shows a plateau, as no more protein binds. At this point, unbound receptors can be washed away. With continued washing, bound receptors now start to dissociate and are removed in the flow of the washing solution (last panel). The sensorgram now shows a declining curve, reflecting the rate at which the receptor and ligand dissociation occurs.